Respiratory Syncytial Virus (RSV) is the major cause of bronchiolitis in infants. Human infant bronchioles infected by RSV are commonly noted to be occluded by sloughed, RSV-infected epithelial cells which along with neutrophil-dominant inflammatory cell infiltrates contribute to acute obstruction of the distal airways. Our long term goals are to understand why RSV has such a propensity for causing bronchiolitis in human infants and to identify therapeutic approaches for reducing the severity of RSV distal airway disease. We have recently identified the RSV Non-Structural protein 2 (NS2) as being responsible for the shedding of RSV-infected columnar epithelial cells providing direct evidence that epithelial cell shedding may be a specific consequence of RSV infection. Using hamsters as an in vivo model of airway infection we have demonstrated RSV NS2-promoted cell shedding into the distal bronchiolar airway lumen results in robust accumulation of shed and virus-infected epithelial cells which clog and obstruct the infected distal airways. As these pathological consequences of RSV NS2 expression in hamster distal airways are strikingly similar to the histopathology obtained from human infants with RSV bronchiolitis we propose RSV NS2 is an important viral genetic determinant for distal airway cytopathology and disease during RSV infection. In this Pilot and Feasibility study, we focus on the development of better models for further understanding the consequences of RSV NS2 expression to enable testing of novel therapies to modulate the cell shedding response. We also attempt to identify novel biomarkers of RSV NS2 expression in the distal airways useful for determining efficacy of novel therapeutic approaches in the lab and in the clinic. Strategies to modify the cell shedding response to RSV NS2 may provide novel therapeutic approaches for reducing the incidence and severity of RSV bronchiolitis. We perform three independent but inter-related Aims: Specific Aim 1: Are the consequences of RSV NS2 expression more prolonged and severe in the distal airways of infant hamsters? Specific Aim 2. Generation of Precision Cut Lung Slices to investigate the impact of RSV NS2 expression in the bronchiolar airway epithelium ex vivo. Specific Aim 3. Does RSV NS2 modification of epithelial keratins provide novel biomarkers for distal airway infection? By performing these Aims we expect to generate better models for understanding the impact of RSV NS2 expression on distal airway pathology and identify novel biomarkers for assessing distal airway epithelium involvement during RSV infection. Successful completion of these Aims will serve as the foundation of future studies to identify strategies to reduce the impact of the consequences of RSV NS2 expression.